Procedures to repair bone loss, fracture non-unions, spinal fusions, total joint replacement, as well as, ruptures to tendon, ligament and muscle affect well over 2 million Americans annually. Surgical repair techniques for all of these procedures have the potential for either incremental or revolutionary improvement through biomaterial nanofiber based strategies. The development of these musculoskeletal tissues from mesenchymal stem cells, MSCs, involves a unique niche composed of assorted extracellular matrix proteins and growth factors. Not surprising, the ECM composition is unique to the musculoskeletal tissue. Cells express approximately 150 different proteins involved in nearly 700 unique interactions, the adhesome, which they use to sense and respond to unique ECM compositions. Of all current proteins in the adhesome, three are directly involved in 36% of all kinase interactions in the adhesome, FAK, Src and Fyn. This proposal seeks to make clear the aspects of adhesion signaling leading to altered phenotype involved with MSC attachment to nanofibers presenting a range of diameters. Understanding how the geometry of the underlying substrate alters the localization and activation of adhesion related proteins will provide design criteria enabling the generation of synthetic MSC niche's capable of directed differentiation down musculoskeletal lineages. Future biomaterial substrates cannot ignore the role that simple aspects such as shape have on directing progenitor cells to the target tissue. This grant seeks to merge the disciplines of regenerative medicine, materials science and cell biology to determine a mechanism by which MSCs sense and respond to the curvature of a nanofiber leading to altered phenotype and ultimately altered lineage commitment down musculoskeletal lineages. Preliminary evidence in support of this grant has indicated that there is a correlation between fiber diameter and focal adhesion size/maturity. The fiber diameters corresponding to the largest adhesions also demonstrated increased RhoA activity and cytoskeletal stiffness. Additionally, nanofiber diameter demonstrated a correlation over MAPK activity, indicating a possible connection to lineage commitment. Specific Aim 1 will produce nanofiber substrates that demonstrate a range of diameters from 1.5m to 500nm; while maintaining consistency with all other geometric parameters of a nanofiber substrate and will examine the binding of FAK to either Src or Fyn. Specific Aim 2 examines the unique phenotype present on each fiber diameter, i.e. migration, proliferation and lineage commitment. Specific Aim 3 seeks to bring together the previous two aims and correlate the nanofiber diameter dependent alterations in FAK/Src-family kinase binding and activation with the altered phenotypes observed. Successful completion of this proposal will provide design guidelines for future biomaterial architectures and advance biology through identification of an intracellular curvature sensing mechanism.